First molecular evidence of potential Culicoides vectors implicated in bluetongue virus transmission in Morocco

Background Bluetongue is a non-contagious viral disease that affects both domestic and wild ruminants. It is transmitted primarily by small hematophagous Diptera belonging to the genus Culicoides (Diptera: Ceratopogonidae). The current study represents the first molecular investigation into the potential role of Culicoides imicola, Culicoides paolae, Culicoides newsteadi, Culicoides spp., and Culicoides circumscriptus as bluetongue virus (BTV) vectors in Morocco. Additionally, the study aimed to evaluate the vectorial activity of midges during the survey seasons. Methods Parous females of these species were captured from several regions of Morocco (6 out of 12) from 2018 to 2021 using Onderstepoort Veterinary Institute (OVI) traps. A total of 2003 parous female specimens were grouped into 55 batches. The midge body of each batch was dissected into three regions (head, thorax, and abdomen), and these regions were analyzed separately using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results BTV RNA was detected in 45 out of the 55 batches tested, indicating a positivity rate of 81.8%. The RT-qPCR-positive pools of the studied Culicoides species exhibited high levels of BTV positivity in each body part (head, thorax, and abdomen), confirming the successful replication of the virus within midge bodies. The BTV circulation was substantial across all three survey seasons (spring, summer, and autumn). High infection rates, calculated using the minimum infection rate (MIR) and maximum likelihood estimation (MLE), were observed during the collection seasons, particularly in autumn and spring, and for all investigated Culicoides species, most notably for C. imicola and C. newsteadi. These increased infection rates underscore the significant risk of Culicoides transmitting the BTV in Morocco. Conclusions The detection of BTV positivity in Culicoides spp. (lacking wing spots that allow their differentiation according to morphological identification keys) suggested that other Culicoides species are competent for BTV transmission in Morocco. The study results indicated, for the first time at the molecular level, that C. imicola and C. newsteadi are the primary potential vectors of BTV in Morocco and that C. paolae and C. circumscriptus are strongly implicated in the propagation of bluetongue at the national level. Graphical Abstract

belongs to the Orbivirus genus of the Reoviridae family, characterized by a double-stranded RNA genome with 10 segments coding for various proteins.Currently, at least 36 different BTV serotypes are circulating worldwide [2][3][4].
A series of events occurs in female midges following ingestion of the virus during their blood meal.These include the liberation of the virus, followed by the infection of the midgut cells, proliferation within these cells, escape from the midgut, spreading into extraintestinal tissues, infection of the salivary glands located between the head and thorax, and finally, transmission of the virus to the subsequent host through the salivary glands.The viral particles must break through various barriers to infection within the abdomen, thorax, and head to successfully disseminate throughout the insect body.These obstacles influence the virus titer and its prevalence during infection, impacting pathogen transmission rates during feeding events.Moreover, the insect's susceptibility or refractoriness to a virus is related to extrinsic factors such as gut microbiota, as well as heritable genetic features like antiviral immune defenses in the insect.These factors are crucial for understanding the vector competence of a midge [18][19][20][21][22][23].
Detection, quantification, and isolation of the virus in field-collected Culicoides midges, analysis of infection levels under natural conditions, and laboratory infection trials are methods for assessing the vectorial competence of Culicoides.The species vectors of BTV have been determined accordingly [24][25][26].Approximately 30 Culicoides species have been associated with BTV transmission, among which C. imicola is recognized as the major vector in Africa [27,28].Due to challenges in conducting studies on Culicoides vector competency, natural infection rates are generally considered a reliable approximation of the vector role [26].
The endemic status of bluetongue in Morocco underscores the importance of identifying Culicoides species vectors of the disease to implement targeted management strategies.Consequently, assessing the infection levels of these species is imperative for a deeper understanding of the transmission dynamics of the disease.
The current study marks the first molecular investigation in Morocco to investigate the potential role of Culicoides imicola, Culicoides paolae, Culicoides newsteadi, Culicoides spp., and Culicoides circumscriptus, collected during entomologic investigations conducted between 2018 and 2021 in different Moroccan regions.The study aimed to assess their involvement in BTV transmission, explore vectorial activity seasons, and investigate their epidemiological contribution and vectorial competence.This was achieved through the detection of virus dissemination in the bodies of field-caught parous females of these species, and the examination of infection rates using two estimates, the minimum infection rate (MIR) and the maximum likelihood estimation (MLE).

Culicoides field collection
Parous females of C. imicola, C. paolae, C. newsteadi, Culicoides spp., and C. circumscriptus were collected using Onderstepoort Veterinary Institute (OVI) light traps across different regions in Morocco (6 out of 12) from 2018 to 2021 (Fig. 1).Species were identified using relevant morphological keys [29,30].The designation Culicoides spp. is assigned to those lacking wing spots, aiding differentiation according to identification keys.The midges were collected in a solution specifically prepared in the laboratory, composed of ethylenediaminetetraacetic acid (EDTA), sodium citrate, ammonium sulfate, and sterile water.This solution was designed to preserve any potential viral RNA that could be present in Culicoides at ambient temperature during the night of capture, from sunset to sunrise.Subsequently, the Culicoides were transferred to 70% ethanol and stored at −20 °C.

Detection of BTV infection in Culicoides midges
A total of 2003 specimens were grouped into 55 pools, each containing no more than 45 midges from the same zone and catch time.
The midges were dissected and separated into three parts: head, thorax, and abdomen.Each body region was collected in a separate subpool.Thus, every pool was subdivided into three subpools.
Viral RNA was isolated from the supernatant obtained by grinding the insect pools in phosphate-buffered saline (PBS) using the MagMAX ™ Viral RNA Isolation Kit (Applied Biosystems) according to the manufacturer's instructions.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were performed using the LSI VetMAX ™ BTV NS3 All Genotypes Kit (Applied Biosystems) in accordance with the manufacturer's instructions.The kit includes two sets of primers/TaqMan probes, one labeled FAM ™ -NFQ (non-fluorescent quencher), encoding segment 10 of BTV RNA, and the other labeled VIC ™ -TAMRA ™ , for the internal positive control.
Reverse transcription and amplification were performed on an Applied Biosystems 7500 Fast Real-Time PCR system with the following thermal cycler program: 45 °C for 10 min, 95 °C for 10 min, followed by 40 cycles of denaturation (95 °C for 15 s) and annealing/extension (60 °C for 45 s).The cycle threshold (Ct) value was defined once the fluorescent signal breached a threshold fluorescence line.An insect pool with a Ct lower than 40 on the FAM-NFQ detector was considered BTV-positive.

Data processing and statistical analysis
QGIS 3.30.1 software was used to construct a map showing the regions of origin of the studied Culicoides species in Morocco.
The data were analyzed using R software (version 4.2.3).Infection rates for Culicoides were calculated using the PooledInfRate package, with a confidence interval (CI) of 95% and a scale of 100.MLE estimate infection rates based on probabilistic models following a binomial distribution.The MIR defines the minimum number of infected individuals per 100 midges tested.Pearson's Chi-square and Fisher's exact tests were used to compare Culicoides pool positivity rates by species and season.The simultaneous application of both tests strengthened the validity of the results.Differences were considered statistically significant at P < 0.05 and a 95% CI.

RT-qPCR results for Culicoides batches
The individual analysis of the three body regions of the specimens within each pool revealed similar results.The Ct values of the head, thorax, and abdomen subpools were similar for each origin pool (Table 1).Consequently, RT-qPCR results are presented based on the pools of origin.
The viral loads of positive pools were significant for all tested species, with Ct < 30 1).

RT-qPCR results for Culicoides pools according to season
The different batches comprised specimens collected during three seasons (spring, summer, and autumn).The analysis resulted in nine Culicoides-positive pools out of 10 collected in spring (90%), 25 out of 32 in summer (78.1%), and 11 out of 13 in autumn (84.6%).
In spring, all tested batches of C. imicola were positive (2/2), as were seven out of eight of C. newsteadi.For the summer pools, four of five C. imicola, 12 of 17 C. paolae, three of four C. circumscriptus, and all Culicoides spp.pools tested (6/6) were positive.In the autumn population, all pools of C. imicola (9) and C. newsteadi (2) were positive, while both batches of Culicoides spp.tested were negative (Fig. 3).

The infection rates of bluetongue virus in Culicoides species (MIR and MLE)
The MIR and MLE are key indicators of Culicoides dynamics in BTV transmission.Assuming that a PCRpositive batch includes at least one infected female, the average MIR for Culicoides was 2.2% (95% CI: 1.6-2.9%).However, MLE directly estimates the proportion of infected insects in the pool, giving an average value of 4.9% (95% CI: 3.3-6.4%).Both MIR and MLE were calculated to understand the distribution of bluetongue within the dominant Culicoides species in Morocco and across the seasons covered by the survey.
The infection rates of Culicoides for three seasons of capture, spring, summer, and autumn, were similarly high (Table 3), ranging from 2.2 to 2.4% for MIR and   Fig. 4 Variations in the infection rates of bluetongue virus (MIR and MLE) in midges tested across species (A) and seasons (B).BTV bluetongue virus; MIR minimum infection rate; MLE maximum likelihood estimation 4.4 to 5.8% for MLE.The highest rates were reached in autumn and spring (Fig. 4B).

Discussion
The parous females of C. imicola, C. paolae, C. newsteadi, Culicoides spp., and C. circumscriptus collected across different regions of Morocco (6 out of 12 Moroccan regions) were screened for BTV RNA using RT-qPCR.The analysis revealed BTV positivity in 45 of the 55 batches tested.The detection of the virus in the head, thorax (containing salivary glands), and abdomen with narrow Ct values (Ct < 30) for each species indicated BTV replication in the body of these species and their potential implication as BTV vectors [25,31].An understanding of the barriers that control the dissemination of BTV within the Culicoides body is essential for studying their competence and susceptibility to infection.Culicoides females ingested approximately 100 TCID50 (50% tissue culture infectious dose; an average of 10 6 TCID50/ml of virus) during a blood meal of typically 10 -4 ml.After feeding, the viral concentration decreased following inactivation of part of the viral particles in the intestine and excretion via the anus.Subsequently, the viral load increased, reaching a plateau of around 5-6 log 10 TCID50 a week later, corresponding to a viral multiplication of 10 3 to 10 4 per midge.The intestinal wall represents the first barrier-the mesenteron infection barrier (MIB)-crossed by the virus.Only the particles remaining in the intestinal lumen can infect intestinal cells.To enter the hemocoel, the virions infecting the intestinal cells must face a second barrier, the mesenteron escape barrier (MEB).Once inside the hemocoel, these viral particles contaminate secondary organs.Before reaching the salivary glands, particles infect adipocytes, representing the third barrier, the dissemination barrier (DB), which can destroy part of the virus.At higher concentrations, viral particles can successfully cross the fourth barrier, the salivary gland infection barrier (SGIB), to penetrate the salivary glands.However, for an insect to infect a new susceptible host, virus particles must also overcome the fifth and final barrier, the salivary gland escape barrier (SGEB), which depends on genetic factors [32,33].The interplay between the virus and the insect's innate immune system, expressed at the barriers, plays a crucial role in the outcome of virus infection and vector survival.The primary antiviral defense mechanism involves the RNA interference (RNAi) pathway, reacting to the detection of the virus-derived double-stranded RNA to suppress its replication.Other innate immune pathways such as Toll, IMD (immune deficiency), and JAK/STAT (Janus kinase/signal transducer and activator of transcription), as well as the autophagy pathway, are also implicated in regulating immune responses in Culicoides.Nonetheless, the midges display refractory and permissive lineages, underlining the heritability of traits linked to their vectorial competence.This indicates that Culicoides exhibit inter-individual variability in vector competence, relating to their transcriptomes and the genomic characteristics of refractory and permissive lineages [21,22,[34][35][36][37]. Accordingly, the mechanisms of virus transmission by Culicoides can differ according to the virus in question and Culicoides species.That is why certain Culicoides species may not express some of these barriers, as observed in Culicoides variipennis, which does not appear to exhibit SGIB or SGEB for BTV [23,38].Indeed, detecting the BTV in the head and thorax after it has crossed several barriers could indicate its successful dissemination within the Culicoides body [20,23].
The calculation of MIR and MLE is essential for assessing the transmission risk of vector-borne diseases.MIR estimates the minimum number of infected vector species among a given sample of vectors, while the MLE accurately estimates the infection prevalence in the vector population.The interpretation of infection rates, especially for low abundance species with small batch sizes, can lead to abnormally high rates.Calculating infection rates using both indices, with MLE considering the size of the tested pools, helps overcome this bias [26].These infection rates can be employed to evaluate the vectorial competence of Culicoides [24].The high infection rates (≥ 1‰) recorded in field populations in the current study suggest a significant risk of BTV transmission in Morocco [39].
The findings revealed active BTV circulation during the survey seasons (spring, summer, and autumn), with increased proportions of positivity.Although the statistical difference in pool positivity between seasons and among species was not statistically significant, it suggests a similar potential for different species to transmit the virus across the three seasons.Nevertheless, the highest MIR and MLE infection rates were observed in autumn and spring.These collection seasons are associated with favorable circumstances for vector activity, thereby increasing the opportunities for BTV transmission [40,41].The roles of C. imicola and C. newsteadi as BTV vectors have previously been identified in the literature [25,[42][43][44][45][46].Their abundance, along with their highest positivity and infectious rates, confirms, for the first time, their role as a primary potential vector of BTV in Morocco.
Culicoides paolae was initially implicated in BTV transmission in Sardinia in 2017, and C. circumscriptus has recently been found BTV-positive in Turkey and Sardinia [42,47].Both species were frequently collected in the current study, and their tested pools exhibited strong positivity for BTV.Higher MIR and MLE values were associated with these Culicoides species, indicating their greater susceptibility to BTV.As a result, this study was the first to highlight the implications of C. paolae and C. circumscriptus as potential vectors of BTV at the national level.
Meanwhile, numerous specimens of Culicoides spp.lacking wing spots and posing identification challenges were captured en masse in this survey.Wing spot patterns are commonly used to identify Culicoides species.However, Culicoides can also be identified based on features other than wing spots.These include microscopic details of the antennae, maxillary palps, and genitalia, as well as morphological characteristics such as body and wing size, the coloration of the dorsum of the thorax, type and number of antennal sensilla, and leg color patterns.This approach is time-consuming and impractical for a large number of individual midges.To address this limitation, molecular methods can be used for the rapid identification of Culicoides species [13,29,[48][49][50][51].Given the frequency of these Culicoides spp. in Morocco, RT-qPCR analysis was carried out on batches of these midges to detect BTV and assess their possible contribution to its spread in the country.The pools exhibited impressive infection rates, highlighting the potential role of these midges in BTV epidemiology.This emphasizes the need for more detailed molecular analysis to identify them, as other Culicoides species could be competent to transmit BTV in Morocco [48,[52][53][54].
Several criteria are required to confirm that an arthropod serves as an arbovirus vector.Firstly, it is necessary to assess the abundance and wide distribution of the suspected arthropod.Secondly, it is imperative to demonstrate that this arthropod has fed on a susceptible host.Furthermore, the virus must be isolated from naturally infected arthropods.It is also important to prove the ability of the arthropod to become infected by feeding either on a viremic host or on a laboratory-infected blood meal.Ultimately, the ability of the infected arthropod to transmit the virus to a susceptible host during hematophagy needs to be confirmed [55][56][57].While BTV infection levels were high and the virus was well-spread in all Culicoides body regions, confirming the competence of the vector requires further validation through virus isolation and experimental infection studies [25].
BTV was detected in small ruminants in the same study regions during the same survey period [7], providing evidence of the active circulation of the virus and the impact of these Culicoides species in bluetongue outbreaks in Morocco.The diversity of BTV-vector Culicoides species in the Moroccan fauna poses a potential threat to livestock herds, reinforcing the importance of implementing effective management strategies and vector control programs.

Conclusions
The current study provided the first molecular evidence illustrating the involvement of C. imicola, C. paolae, C. newsteadi, Culicoides spp., and C. circumscriptus as potential vectors for BTV propagation at the national level, along with insights into the vector activity seasons of these insects.BTV detection in parous females of field species, after the virus has crossed multiple body barriers, and the analysis of infection rates (MIR and MLE) for these midge populations served to assess the vectorial competence of Culicoides.Indeed, the results confirmed the implication of the studied species (C.imicola, C. paolae, C. newsteadi, Culicoides spp., and C. circumscriptus) as potential BTV vectors in bluetongue epidemics in Morocco, with active virus circulation during spring, summer, and autumn.
analyzed and interpreted the data, prepared the maps, performed the statistical analysis, produced all the figures, and wrote the manuscript.OFF and IEB revised the manuscript.All the authors have read and approved the final manuscript.

Fig. 1
Fig. 1 Regions of origin for Culicoides species investigated in the Kingdom of Morocco from 2018 to 2021

Fig. 2 Fig. 3
Fig. 2 Batches of Culicoides species tested for bluetongue virus by RT-qPCR . Ct values for positive pools of C. imicola ranged between 19.6 and 29.5, C. paolae between 24.7 and 29, C. newsteadi between 18.4 and 29.8, Culicoides spp. between 22 and 25.8, and C. circumscriptus between 21.1 and 26.6.The highest virus loads were observed in the head, thorax, and abdomen of C. imicola and C. newsteadi (Ct < 20) (Table

Table 1
Average Ct values of various body parts in parous Culicoides females collected in Morocco (6 out of 12 regions) from 2018 to 2021

Table 2
Infection rates of bluetongue virus in parous Culicoides females captured in Morocco MIR minimum infection rate; MLE maximum likelihood estimation; CI confidence interval

Table 3
Infection rates of bluetongue virus in parous Culicoides females according to collection season MIR minimum infection rate; MLE maximum likelihood estimation; CI confidence interval